A dietary fibre prepared from sugarcane stalk was compared with psyllium husk and wheat dextrin. In contrast to the other dietary fibres, sugarcane fibre was found to contain significant amounts of insoluble dietary fibre (73–86%), lignin (18.66–20.23%), and rare minerals such as chromium (0.67–2.54 mg/100 g) and manganese (1.07–2.34 mg/100 g). Analysis of the ethanol extract also detected compounds with antioxidant activity. Characterisation of five sugarcane fibres prepared from selected strains, harvest periods (growth or storage phase), and processing conditions showed these factors influenced the final composition. Furthermore, using in vitro digestion, we found that potassium, magnesium, chromium, and zinc in were bioaccessible in sugarcane samples. Also, sodium was shown to bind to the sugarcane fibre potentially indicating bile salt binding activity. Results from this study support the use of sugarcane as a source of dietary fibre in functional foods.